The present invention relates to the field of automated biological analysis systems and more specifically to automated cell detection systems employing waveguides having photosensitive components.
The automated detection of rare cells in a population of different cells is a challenging problem akin to finding the proverbial needle in the haystack. Examining cell samples using traditional microscopy would require unreasonable amounts of time and is susceptible to operator error. Furthermore, in some instances, for example, the detection of microbial cells in natural water ecosystems, the characteristics of the rare cells are not already known. In such situations optical microscopy based on image processing methods is the only viable alternative. A similar approach may be needed when the cells in question are known, but they are not identifiable through any other mechanism except with traditional microscopy and image processing, e.g. in the detection of cancer cells in papsmears.
In cases where a specific characteristic of the rare cell can be identified and located through the generation of a distinct signal, detection methods other than image processing may be employed allowing for much faster detection in a very large initial population of cells. If, for example, a cell surface antibody specific to the cell in question can be bound to a fluorescing substance, the cell can be detected using fast methods such as fluorescence activated cell sorting (FACS). In such systems though the efficiency of detection is inversely proportional to the frequency of the cells in question.
U.S. Pat. No. 4,746,179 issued on May 24, 1988 to Dahne et al. describes the use of a waveguide in conjunction with a fluorescence signal generated by the sample to estimate the concentration of a soluble antigen. Dahne et al. passes an excitation signal through the waveguide that is immersed in, or in direct contact with, the sample solution. The leakage of the excitation signal from the waveguide reacts with the solution next to the waveguide and produces a fluorescence signal that is picked up by the waveguide and directed to and measured by a detector. The strength of the fluorescence signal will be proportional to the concentration of the sample in solution.
Citation or identification of any reference in this section or any section of this application shall not be construed that such reference is available as prior art to the present invention.
This present invention has for its objects devices, systems, and methods for identification of particles of interest in low concentrations and with high accuracy. Not only are particles of interest identified, but also their spatial position in a measurement region may be identified, permitting later verification by means of microscopic image processing. Although the present invention may be used to detect the presence and position of a wide range of particles, its primary application is to cell detection, wherein it improves on current methods of cell screening. In the following, but without limitation, the invention will be primarily described in its principal application.
The present invention achieves its objects by means of a novel and inventive combination of a two-dimensional (2D), photosensitive waveguide with a 2D scanning of the waveguide with the intersection of beams of radiation. In a photosensitive waveguide, the properties of guided radiation change in response to incident light. The photosensitive waveguides of the present invention are based on novel uses of nonlinear optical (NLO) effects that take place in certain substances. These effects result in changes of optical properties, such as index of refraction or absorptivity, in response to the intensity of the incident light. These substances can be various organic compounds e.g. conjugated dye molecules or special polymers. Such a compound may be used as a cladding for a cylindrical or a flat optical waveguide. A biological specimen containing various kinds of cells can be treated so that certain cells emit radiation to which the optical properties of the cladding are responsive. If the cells are juxtaposed to the guide, it will affect the attributes of light radiation transferred by the waveguide. By monitoring the changes in the properties of the light transferred through the waveguide, one can detect the presence of the interesting cells.
This invention also provides for determining the location of interesting cells in a largely 2D measurement region by making use of the above detection principle. If two non-parallel beams are guided through a waveguide, both will have detectable changes if an emitting cell is in proximity to the intersection of the two beams. Thus, the presence and position of labeled cells can be determined by scanning the region of intersection of two non-parallel beams guided through the waveguide. In various alternatives, the waveguide may be moved or a beam of the fluorescence-inducing radiation can be scanned, or both.
In the following, a cell emitting radiation that controls a photosensitive waveguide will cause detectable changes in the properties of a light beam that is guided in proximity to the emitting cell. Typically, the necessary proximity is within one to three cell diameters.
In more detail, the present invention has several embodiments. In a first embodiment the invention includes a device for detecting the presence of one or more particles, wherein the particles emit controlling radiation and are placed in a measurement region, the device comprising: a photosensitive waveguide, wherein one or more properties of radiation guided through the waveguide are responsive to controlling radiation emitted by particles present in the measurement region, and a photo-detection system responsive to the one or more properties of the radiation guided through the photosensitive waveguide, wherein particles emitting controlling radiation in the measurement region cause changes in the one or more properties of the radiation guided through the waveguide which are detectable by the photo-detection system, whereby the system detects the presence of particles.
In a second embodiment the invention includes a system for detecting the presence and position of one or more cells which emit controlling radiation comprising: a measurement region in which the cells are affixed, a two-dimensional (2D) photosensitive waveguide, wherein one or more properties of radiation guided through the waveguide are responsive to controlling radiation emitted by cells present in the measurement region, and a photo-detection system responsive to the one or more properties of a first beam of radiation and of a second beam of radiation, wherein the first and second beam are guided through the photosensitive waveguide in non-parallel directions, wherein cells emitting controlling radiation in the measurement region cause changes in the one or more properties of the first or the second beam of radiation when the beams guided through the waveguide in proximity to an emitting cell, the changed properties being detectable by the photo-detection system, whereby the system the presence and position of cells.
A first aspect of the second embodiment further includes means for moving the first and the second beam of radiation in non-parallel directions so that their region of intersection scans substantially all of the 2D photosensitive waveguide that is exposed to the measurement region, and a controller for providing control signals to the photo-detection system and to the means for moving. Further, the controller may include a memory, and a processor coupled to the memory and for causing the generation of the control signals, wherein the memory contains encoded program instructions for causing the processor to perform the steps of (i) generating control signals to cause the means for moving to move the first and the second beam of radiation so that their region of intersection scans substantially all of the measurement region, (ii) generating control signals to cause the photo-detection system to detect the changed properties of the beams, (iii) storing the positions of the beams when the photo-detection system detects changed properties, and (iv) computing the presence and position of cells from the stored positions.
In a third embodiment the invention includes a system for detecting the presence and position of one or more cells, wherein the cells are labeled to emit controlling radiation in response to incident activation radiation, the system comprising: a two-dimensional (2D) photosensitive waveguide, wherein one or more properties of radiation guided through the waveguide are responsive to controlling radiation emitted by cells present in the measurement region, and wherein the waveguide is planar and substantially disk shaped, and a measurement region in which the cells are affixed, means for rotating the disk-shaped 2D planar photosensitive waveguide together with the measurement region, a photo-detection system responsive to the one or more properties of a beam of radiation, wherein the beam is guided through the photosensitive waveguide along a diameter of the disk-shaped 2D planar photosensitive waveguide, and means for scanning a beam of activation radiation along the path of the beam guided though the waveguide, wherein the activation radiation causes the labeled cells to emit controlling radiation, wherein cells emitting controlling radiation in the measurement region in response to incident activation radiation cause changes in the one or more properties of the beam of radiation when the beam guided through the waveguide in proximity to an emitting cell, the changed properties being detectable by the photo-detection system, whereby the system the presence and position of cells.
A first aspect of the third embodiment further includes a controller for providing control signals to the means for rotating and to the means for scanning activation radiation, where the controller further includes a memory, and a processor coupled to the memory and for causing the generation of the control signals, wherein the memory contains encoded program instructions for causing the processor to perform the steps of (i) generating control signals to cause the means for rotating and the means-for scanning so that the region of intersection of the beam guided through the waveguide and the activation beam scans substantially all of the measurement region, (ii) generating control signals to cause the photo-detection system to detect the changed properties of the beam guided through the waveguide, (iii) storing the angular position of the waveguide and the position of the beam of activation radiation when the photo-detection system detects changed properties, and (iv) computing the presence and position of cells from the stored positions.
In a fourth embodiment the invention includes a method for determining the presence and position of one or more cells which emit controlling radiation comprising: affixing the cell in a measurement region, wherein controlling radiation emitted in the measurement regions is incident on a two-dimensional (2D) photosensitive waveguide, and wherein one or more properties of radiation guided through the waveguide are responsive to controlling radiation emitted by cells present in the measurement region, guiding two or more beams of radiation through the 2D photosensitive waveguide at a series of positions so that the intersection of the beams scans substantially all of the 2D photosensitive waveguide illuminated by the measurement region, detecting the one or more properties of the beams guided through the waveguide, wherein presence and position of emitting cells is determined as the proximity of intersection of the beams when changed properties of the beams are detected.
In a fifth embodiment the invention also includes computer readable media comprising the encoded program instruction for the controllers of the invention.
In all embodiments it is preferable that the cells being detected are labeled with a fluorophore, and that the embodiment further comprise a source for activation radiation incident on the measurement region for stimulating the fluorophore to fluoresce. Then, this fluorescence is the controlling radiation.